New polyhydroxyalkanoate comprising unit having (phenylmethyl) oxy structure on side chain thereof, and method for producing the same

ABSTRACT

Provided are a PHA having an active group, which is produced by microorganisms with high productivity, and in which the ratio of units on its side chain having an active group can be controlled and the physical properties can be arbitrarily controlled so that its application as a polymer is not limited, and a method for producing the same. 
 
A 3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acid unit expressed by the following chemical formula (1):  
                 
wherein x can be one or more integers within the range shown in the chemical formula.

TECHNICAL FIELD

The present invention relates to a polyhydroxyalkanoate comprising a newunit, and a method for producing the same using microorganisms.Hereinafter, the term “polyhydroxyalkanoate” is referred to as “PHA” attimes.

BACKGROUND ART

It has been reported so far that many types of microorganisms producepoly-3-hydroxybutyric acid (PHB) or other polyhydroxyalkanoates (PHAs)and accumulate them in the cells. As with the conventional plastics,polymers such as polyhydroxyalkanoate produced by microorganisms aresubjected to melting processing, so that they can be used for productionof various types of products. Moreover, polymers produced bymicroorganisms, such as polyhydroxyalkanoates, are biodegradable, andaccordingly, they have an advantage in that they are completelydecomposed by microorganisms existing in the nature. Accordingly, forexample, when a polyhydroxyalkanoate produced by microorganisms isdiscarded, differing from many conventional synthetic polymer compounds,it does not remain in the environment as is, and therefore it does notcause pollution. Furthermore, since polyhydroxyalkanoate produced bymicroorganisms have excellent biocompatibility, it is expected thatthese compounds will be applied to soft components for medical use, etc.

It is known that these polyhydroxyalkanoates produced by microorganismscan have various compositions or structures depending on the type ofmicroorganisms used for production, the composition of a medium, cultureconditions, etc. Studies have been made so far to attempt to control thecomposition or structure of the polyhydroxyalkanoate produced bymicroorganisms, mainly from the viewpoint of the improvement of physicalproperties of the polyhydroxyalkanoate.

As a study directed towards controlling the composition or structure ofthe polyhydroxyalkanoate produced by microorganisms, a study has beenvigorously made to allow microorganisms to produce polyhydroxyalkanoatehaving an aromatic ring in its unit in these years.

(a) Polyhydroxyalkanoate Comprising Phenyl Group or its PartiallySubstituted Form

It has been reported that using 5-phenyl valeric acid as a substrate,Pseudomonas oleovorans produces a polyhydroxyalkanoate comprising3-hydroxy-5-phenyl valeric acid as a unit (Makromol. Chem., Vol. 191,1990, pp. 1957-1965, Macromolecules, Vol. 24, 1991, pp. 5256-5260).

It has been reported that using 5-(p-tolyl)valeric acid as a substrate,Pseudomonas oleovorans produces a polyhydroxyalkanoate comprising3-hydroxy-5-(p-tolyl)valeric acid as a unit (Macromolecules, Vol. 29,1996, pp. 1762-1766).

It has been reported that using 5-(2,4-dinitrophenyl)valeric acid as asubstrate, Pseudomonas oleovorans produces a polyhydroxyalkanoatecomprising 3-hydroxy-5-(2,4-dinitrophenyl)valeric acid and3-hydroxy-5-(p-nitrophenyl)valeric acid as units (Macromolecules, Vol.32, 1999, pp. 2889-2895).

(b) Polyhydroxyalkanoate Comprising Phenoxy Group or its PartiallySubstituted Form

It has been reported that using 11-phenoxy undecanoic acid as asubstrate, Pseudomonas oleovorans produces a polyhydroxyalkanoatecopolymer comprising a 3-hydroxy-5-phenoxy valeric acid unit and a3-hydroxy-9-phenoxynonanoic acid unit (Macromol. Chem. Phys., Vol. 195,1994, pp. 1665-1672).

There has been disclosed an invention relating to a homopolymerconsisting of 3-hydroxy-5-(monofluorophenoxy)pentanoate (3H5(MFP)P)units or 3-hydroxy-5-(difluorophenoxyl)pentanoate (3H5(DFP)P) units; acopolymer containing at least (3H5(MFP)P) units or (3H5(DFP)P) units;Pseudomonas putida having an ability to synthesize these polymers; and amethod of producing the above described polymers using Pseudomonasspecies. In addition, it is described as an advantage of the aboveinvention that a long chain aliphatic acid having substituent groups canbe assimilated to synthesize a polymer having a phenoxy groupsubstituted with 1 or 2 fluorine atoms at the side chain terminal andthat the polymer provides stereoregularity and water repellency, whilemaintaining a high melting point and good processability (JapanesePatent No. 2989175).

Moreover, studies are conducted on a polyhydroxyalkanoate in which acyano or nitro group is substituted on an aromatic ring in its unit, aswell as on fluorine-substituted PHA in which fluorine is substituted onan aromatic ring in its unit.

It has been reported that a polyhydroxyalkanoate containing3-hydroxy-6-(p-cyanophenoxy)hexanoic acid or3-hydroxy-6-(p-nitrophenoxy)hexanoic acid as a monomer unit is producedwith octanoic acid and 6-(p-dyanophenoxy)hexanoic acid or6-(p-nitrophenoxy)hexanoic acid as substrates, using a Pseudomonasoleovorans ATCC 29347 strain and a Pseudomonas putida KT 2442 strain(Can. J. Microbiol, Vol. 41, 1995, pp. 32-43 and Polymer International,Vol. 39, 1996, pp. 205-213).

Such a polyhydroxyalkanoate containing units each having an aromaticring having a substituent group thereof can be a multifunctionalpolyhydroxyalkanoate, which possesses a new function derived from thesubstituent group existing on the aromatic ring, while maintainingpolymer characteristics derived from the aromatic ring, such as a highglass transition temperature and good processability.

At the same time, studies are vigorously conducted directed towards theobtainment of a multifunctional polyhydroxyalkanoate, which is based ona polyhydroxyalkanoate having a bromo group in its unit and obtained byintroducing any given functional group into the side chain of a producedpolymer by chemical transformation using the above bromo group.

It has been reported that a polyhydroxyalkanoate having a bromo group ona side chain thereof is produced using Pseudomonas oleovorans, and thenthe side chain is modified with the thiolated product of an acetylatedmaltose, to synthesize a polyhydroxyalkanoate having differentsolubility and hydrophilicity (Macromol. RapidCommun., Vol. 20, 1999,pp. 91-94).

It has been reported that polyester having a vinyl group on a side chainthereof is produced using Pseudomonas oleovolans, and then the vinylgroup in the polyester molecule is oxidized, so as to produce polyesterhaving an epoxy group on its side chain (Polymer, Vol. 41, 2000, pp.1703-1709).

It has been reported that polyester having a vinyl group on a side chainthereof is produced using Pseudomonas oleovolans, and then the vinylgroup is epoxidized, to produce polyester having an epoxy group on itsside chain (Macromolecules, Vol. 31, 1998, pp. 1480-1486).

It has been reported that using a vinyl group on the side chain ofpolyester, a crosslinking reaction is carried out in the polyestermolecule, to improve the properties of the polyester (Polymer, Vol. 35,1994, pp. 2090-2097).

To change the physical properties of a PHA having an active group in itsunit to actually use it as a polymer, the synthesis of a PHA copolymercomprising units other than units having active groups by usingmicroorganisms has been studied. It has been reported that usingPseudomonas oleovorans, a PHA copolymer comprising a3-hydroxy-ω-bromoalkanoic acid unit and a straight-chain alkanoic acidunit has been produced in the coexistence of ω-bromoalkanoic acid suchas 11-bromoundecanoic acid, 8-bromooctanoic acid and 6-bromohexanoicacid and n-nonanoic acid (Macromolecules, Vol. 25, 1992, pp. 1852-1857).

Thus, into PHA having an active group with high reactivity, such as abromo or vinyl group, in its units, various functional groups can beintroduced. Or, chemical transformation can also be performed on suchPHA. Moreover, since PHA having an active group can be a crosslink pointof a polymer, it can be said that such PHA is extremely effective forachievement of multifunctional PHA.

However, in a case where a PHA having a bromo group as an active groupis synthesized using microorganisms, the productivity of the obtainedPHA is low. In a case where a PHA copolymer is synthesized usingmicroorganisms, it is difficult to increase or control the unit ratio ofbromo groups.

Further, in the case of the synthesis of a PHA having a vinyl group asan active group, if the vinyl group is located at the end of an alkylchain, the synthesized PHA has a low glass transition temperature and alow melting point, and therefore it cannot be said that the obtained PHAhas physical properties preferable for the processability and usabilityof the polymer.

For the above described reasons, a new PHA having an active group, whichis produced by microorganisms with high productivity, and in which theratio of units in its side chain having an active group can becontrolled and the physical properties can be arbitrarily controlled sothat its application as a polymer is not limited, and a method forproducing the same, have been desired.

DISCLOSURE OF THE INVENTION

As a result of intensive studies directed towards achieving the aboveobject, the present inventors have found a method for synthesizing a PHAcomprising a unit having a highly reactive (phenylmethyl)oxy structureas an active group by using microorganisms, thereby completing thepresent invention.

According to an aspect of the present invention, there is provided apolyhydroxyalkanoate comprising a3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acid unit expressed by chemicalformula (1):

wherein x can be one or more integers within the range shown in thechemical formula.

According to another aspect of the present invention, there is provideda method for producing a polyhydroxyalkanoate comprising, in a moleculethereof, a 3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acid unit expressedby chemical formula (1), which comprises allowing a microorganism withan ability to produce a polyhydroxyalkanoate comprising in a moleculethereof a 3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acid unit expressed bychemical formula (1) to biosynthesize the polyhydroxyalkanoate under acondition which comprise ω-[(phenylmethyl)oxy]alkanoic acid expressed bychemical formula (19):

wherein x can be one or more integers within the range shown in thechemical formula.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a ¹H-NMR spectrum chart of the polyhydroxyalkanoate in Example1; and

FIG. 2 is a ¹H-NMR spectrum chart of the polyester obtained in Example23.

BEST MODE FOR CARRYING OUT THE INVENTION

The method for producing the polyhydroxyalkanoate of the presentinvention comprises culturing microorganism in a medium containing atleast one selected from a group consisting of peptides, yeast extract,organic acids or salts thereof, amino acids or salts thereof,saccharides, straight-chain alkanoic acids, which is saturated orunsaturated fatty acid having 4 to 12 carbon atoms or salts thereof, inaddition to the ω-[(phenylmethyl)oxy]alkanoic acid expressed by theabove chemical formula (19). Moreover, in the above culture ofmicroorganisms, the peptide contained in the medium is polypeptone; theorganic acids or salts thereof contained in the medium are one or morecompounds selected from a group consisting of pyruvic acid, oxaloaceticacid, citric acid, isocitric acid, ketoglutaric acid, succinic acid,fumaric acid, malic acid, lactic acid, and salts thereof; the aminoacids or salts thereof contained in the medium are one or more compoundsselected from a group consisting of glutamic acid, asparaginic acid, andsalts thereof; and the saccharides contained in the medium are one ormore compounds selected from a group consisting of glyceraldehyde,erythrose, arabinose, xylose, glucose, galactose, mannose, fructose,glycerol, erythritol, xylitol, gluconic acid, glucuronic acid andgalacturonic acid, maltose, sucrose and lactose.

The polyhydroxyalkanoate of the present invention preferably furthercomprises at least one unit expressed by chemical formula selected fromthe group consisting of chemical formulas (2) and (3):

wherein y and z can be one or more integers within the range shown inthe chemical formulas, while being independent from the unit expressedby chemical formula (1).

Alternatively, the PHA of the present invention preferably comprises, ina molecule thereof, the 3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acidunit expressed by chemical formula (1) and a 3-hydroxy-alkanoic acidunit expressed by chemical formula (4):

wherein m can be one or more integers within the range shown in thechemical formula, and R comprises a residue having either a phenylstructure or thienyl structure, or a 3-hydroxy-ω-cyclohexylalkanoic acidunit expressed by chemical formula (5):

wherein R₁ is H, CN, NO₂, halogen, CH₃, C₂H₅, C₃H₇, CF₃, C₂F₅ and C₃F₇,and k can be one or more integers within the range shown in the chemicalformula. R in chemical formula (4) is preferably a group selected fromthe group consisting of

wherein R₂ is H, halogen, CN, NO₂, CH₃, C₂H₅, C₃H₇, CH═CH₂, COOR₃(wherein R₃ represents any one selected from the group consisting of H,Na and K), CF₃, C₂F₅ and C₃F₇, and in a case where there exist aplurality of units, R₂ may be different for each unit;

wherein R₄ is selected from the group consisting of H, halogen, CN, NO₂,CH₃, C₂H₅, C₃H₇, SCH₃, CF₃, C₂F₅ and C₃F₇, and in a case where thereexist a plurality of units, R₄ may be different for each unit;

wherein R₅ is selected from the group consisting of H, halogen, CN, NO₂,CH₃, C₂H₅, C₃H₇, CF₃, C₂F₅ and C₃F₇, and in a case where there exist aplurality of units, R₅ may be different for each unit;

wherein R₆ is selected from the group consisting of H, halogen, CN, NO₂,COOR₇, SO₂R₈ (wherein R₇ represents any one selected from the groupconsisting of H, Na, K, CH₃ and C₂H₅, and R₈ represents any one selectedfrom the group consisting of OH, ONa, OK, halogen, OCH₃ and OC₂H₅), CH₃,C₂H₅, C₃H₇, (CH₃)₂—CH, and (CH₃)₃—C, and in a case where there exist aplurality of units, R₆ may be different for each unit;

wherein R₉ represents a substituent group on the aromatic ring, R₉ isselected from thg group consisting of H, halogen, CN, NO₂, COOR₁₀,SO₂R₁₁ (wherein R₁₀ represents any one selected from the groupconsisting of H, Na, K, CH₃ and C₂H₅, and R₁, represents any oneselected from the group consisting of OH, ONa, OK, halogen, OCH₃ andOC₂H₅), CH₃, C₂H₅, C₃H₇, (CH₃)₂—CH and (CH₃)₃—C, and in a case wherethere exist a plurality of units, R₉ may be different for each unit;

wherein R₁₂ is selected from thg group consisting of H, halogen, CN,NO₂, COOR₁₃, SO₂R₁₄ (wherein R₁₃ represents any one selected from thegroup consisting of H, Na, K, CH₃ and C₂H₅, and R₁₄ represents any oneselected from the group consisting of OH, ONa, OK, halogen, OCH₃ andOC₂H₅), CH₃, C₂H₅, C₃H₇, (CH₃)₂—CH and (CH₃)₃—C, and in a case wherethere exist a plurality of units, R₁₂ may be different for each unit;and

wherein R₁₅ is selected from the group consisting of H, halogen, CN,NO₂, COOR₁₆, SO₂R₁₇ (wherein R₁₆ represents any one selected from thegroup consisting of H, Na, K, CH₃ and C₂H₅, and R₁₇ represents any oneselected from the group consisting of OH, ONa, OK, halogen, OCH₃ andOC₂H₅), CH₃, C₂H₅, C₃H₇, (CH₃)₂—CH and (CH₃)₃—C, and in a case wherethere exist a plurality of units, R₁₅ may be different for each unit.

In the PHA of the present invention, the3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acid unit expressed by chemicalformula (1) is preferably either one or both of:

-   a 3-hydroxy-4-[(phenylmethyl)oxy]butyric acid unit expressed by    chemical formula (6):    and a 3-hydroxy-5-[(phenylmethyl)oxy]valeric acid unit expressed by    chemical formula (7):

In the PHA of the present invention, a number average molecular weightis preferably within the range between 1,000 and 1,000,000.

In the method for producing a polyhydroxyalkanoate of the presentinvention, the polyhydroxyalkanoate preferably comprises at least oneunit expressed by chemical formulas (2) and (3).

In the method for producing a polyhydroxyalkanoate of the presentinvention, the ω-[(phenylmethyl)oxy]alkanoic acid expressed by thechemical formula (19) is preferably 4-[(phenylmethyl)oxy]butyric acidexpressed by chemical formula (23):

or 5-[(phenylmethyl)oxy]valeric acid expressed by chemical formula (24):

The method for producing a polyhydroxyalkanoate of the present inventionmay comprise allowing the microorganism with an ability to produce apolyhydroxyalkanoate comprising, in a molecule thereof, the3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acid unit expressed by chemicalformula (1) and

-   a 3-hydroxy-alkanoic acid unit expressed by chemical formula (22):    wherein m can be one or more integers within the range shown in the    chemical formula, and R₁₈ comprises a residue having either a phenyl    structure or thienyl structure, or a 3-hydroxy-ω-cyclohexylalkanoic    acid unit expressed by chemical formula (5), from    ω-[(phenylmethyl)oxy]alkanoic acid expressed by chemical formula    (19), and alkanoic acid expressed by chemical formula (20):    wherein q can be one or more integers within the range shown in the    chemical formula, and R₁₆ comprises a residue having either a phenyl    structure or thienyl structure-   or ω-cyclohexylalkanoic acid expressed by chemical formula (21)    wherein R₁₇ is selected from the group consisting of H, CN, NO₂,    halogen, CH₃, C₂H₅, C₃H₇, CF₃, C₂F₅ and C₃F₇, and r can be one or    more integers within the range shown in the chemical formula as raw    materials to biosynthesize the polyhydroxyalkanoate under a    condition which comprise ω-[(phenylmethyl)oxy]alkanoic acid    expressed by chemical formula (19), and alkanoic acid expressed by    chemical formula (20) or a ω-cyclohexylalkanoic acid expressed by    chemical formula (21). In particular, R₁₆ in chemical formula (20)    and R₁₈ in chemical formula (22) are preferably groups independently    selected from the group consisting of chemical formula (25):    wherein R₁₉ is selected from the group consisting of H, halogen, CN,    NO₂, CH₃, C₂H₅, C₃H₇, CH═CH₂, CF₃, C₂F₅ and C₃F₇, and in a case    where there exist a plurality of units, R₁₉ may be different for    each unit; chemical formulae (9), (10), (11), (12), (13), (14),    (15), (16) and (17). Alternatively, the condition is preferably that    the microorganism is cultured in a medium containing the    ω-[(phenylmethyl)oxy]alkanoic acid expressed by chemical    formula (19) and the alkanoic acid expressed by chemical    formula (20) or the α-cyclohexylalkanoic acid expressed by chemical    formula (21).

In the method for producing a polyhydroxyalkanoate of the presentinvention, the condition is preferably that the microorganisms iscultured in a medium containing ω-[(phenylmethyl)oxy]alkanoic acidexpressed by chemical formula (19). In particular, the medium preferablycontains at least one selected from the group consisting of peptides,yeast extract, organic acids or salts thereof, amino acids or saltsthereof, saccharides and straight-chain alkanoic acids, which issaturated or unsaturated fatty acid having 4 to 12 carbon atoms or saltsthereof. More especially, the peptide is polypeptone; the organic acidsor salts thereof are one or more compounds selected from the groupconsisting of pyruvic acid, oxaloacetic acid, citric acid, isocitricacid, ketoglutaric acid, succinic acid, fumaric acid, malic acid, lacticacid, and salts thereof; the amino acids or salts thereof are one ormore compounds selected from the group consisting of glutamic acid,aspartic acid, and salts thereof; and the saccharides are one or morecompounds selected from the group consisting of glyceroaldehyde,erythrose, arabinose, xylose, glucose, galactose, mannose, fructose,glycerol, erythritol, xylitol, gluconic acid, glucuronic acid andgalacturonic acid, maltose, sucrose and lactose.

When cultivating the microorganism in the medium containing the alkanoicacid expressed by chemical formula (19) in the method for producing apolyhydroxyalkanoate of the present invention, the culture ofmicroorganisms preferably comprises two or more culturing steps. Inparticular, the culture is preferably a fed-batch culture.

When cultivating the microorganism in the medium containing the alkanoicacid expressed by chemical formula (19) in the method for producing apolyhydroxyalkanoate of the present invention as mentioned above, themethod preferably comprises a step of recovering a polyhydroxyalkanoatecomprising 3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acid unit expressedby chemical formula (1) generated by the microorganism from the cells ofthe microorganism. In the method for producing a polyhydroxyalkanoate ofthe present invention, the microorganism preferably belongs toPseudomonas species. In particular, the microorganism is preferably oneor more strains selected from the group consisting of Pseudomonascichorii YN2 (FERM BP-7375), Pseudomonas cichorii H45 (FERM BP-7374) andPseudomonas jessenii P161 (FERM BP-7376).

Detailed culture conditions of microorganisms in the method forproducing the polyhydroxyalkanoate of the present invention are asfollows:

As described below, various necessary substrates and nutrients are addedto an inorganic salt medium basically containing a phosphate buffer andammonium salts or nitrates.

In order to produce a polyhydroxyalkanoate of interest expressed by theabove chemical formula (1) comprising a3-hydroxy-ω-(phenylmethyl)alkanoic acid unit, it is desirable thatω-[(phenylmethyl)oxy]alkanoic acid expressed by the above chemicalformula (19) is contained in the medium as a substrate, at a proportionfrom 0.01% to 1% (w/v) per medium, and more preferably at a proportionfrom 0.02% to 0.2% per medium.

In order to produce a polyhydroxyalkanoate comprising in a moleculethereof a 3-hydroxy-alkanoic acid unit expressed by chemical formula(22) or a 3-hydroxy-ω-cyclohexylalkanoic acid unit expressed by chemicalformula (5) as well as a 3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acidunit, it is desirable that each of ω-[(phenylmethyl)oxy]alkanoic acid ofthe above chemical formula (19) and an alkanoic acid expressed bychemical formula (20) or ω-cyclohexylalkanoic acid expressed by theabove chemical formula (21) is contained as a substrate in the medium ata proportion from 0.01% to 1% (w/v) per medium, and more preferably at aproportion from 0.02% to 0.2% per medium.

A carbon source and a nitrogen source for growth of microorganisms andfor production of a polyhydroxyalkanoate are preferably added to themedium at a concentration from 0.1% to 5% (w/v) per medium, and morepreferably from 0.2% to 2% per medium.

Any medium can be used in the present invention, as long as it is anorganic salt medium containing phosphate and a nitrogen source such asammonium salts or nitrates. It is possible to increase the productivityof PHA by controlling the concentration of the nitrogen source.

Any temperature is applied as a culture temperature, as long as theabove cell strains can grow favorably at the temperature. A temperaturebetween 15° C. and 37° C. is appropriate, and a temperature between 20°C. and 30° C. is more preferable.

Any culture method can be used, as long as microorganisms can grow andproduce PHA. Not only one stage culture such as usual batch culture butalso two stage culture comprised of the steps of collecting once cellsobtained by a one stage culture and adding the collected cells toanother new medium to carry out a cultivation again can be alsoemployed. A fed-batch culture, in which a new medium is added to theculture liquid without collecting the cells, can be used so as to carryout the two stage culture more simply. A continuous culture also can beused. Further, any form of the culture can be used, including a methodof shaking a medium in a vessel for cultivation such as flask, a methodwith a fermenter and so forth.

In addition to the above described methods, there is another method forallowing microorganisms to produce and accumulate PHA. This methodcomprises making microorganism sufficiently grow, transferring the cellsto a medium in which a nitrogen source such as ammonium chloride islimited, and further culturing them in a state where a compound as asubstrate of a unit of interest is present. This method improvesproductivity in some cases.

Moreover, after the microorganisms are cultured under the abovedescribed conditions, the method may comprise a step of recovering apolyhydroxyalkanoate comprising the3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acid unit expressed by the abovechemical formula (1) produced by the above microorganisms from thecells.

As a method of recovering a PHA of interest from the cells ofmicroorganisms, a common method can be adopted. For example, extractionwith an organic solvent such as chloroform, dichloromethane,ethylacetate or acetone is the most simple, but dioxane, tetrahydrofuranor acetonitrile may also be used in some cases. In an environment inwhich the use of organic solvents is not preferred, any one of atreatment with surfactants such as SDS, a treatment with an enzyme suchas lysozyme, a treatment with chemicals such as hypochlorite, ammoniumor EDTA, an ultrasonic crushing method, a homogenizer method, a pressurecrushing method, a bead impulse method, a grinding method, an immersionmethod and a freeze-thaw method may be used to physically crushmicroorganism cells. Cell components other than PHA are removed by anyone of the above methods, to collect PHA.

As microorganisms used for the production method of the presentinvention, any species of microorganisms may be used, as long as theyhave an ability to satisfy the above described conditions. Among them,microorganisms belonging to Pseudomonas species are desirable. Specificexamples of preferred species include Pseudomonas cichorii, Pseudomonasputida, Pseudomonas fluorecense, Pseudomonas oleovorans, Pseudomonasaeruginosa, Pseudomonas stutzeri, and Pseudomonas jessenii. Morespecifically, examples of a suitable strain include Pseudomonas cichoriiYN2 (FERM BP-7375), Pseudomonas cichorii H45 (FERM BP-7374), andPseudomonas jessenii P161 (FERM BP-7376). These three types of strainswere deposited on Nov. 20, 2000 at the International Patent OrganismDepositary (IPOD) of National Institute of Advanced Industrial Scienceand Technology (AIST), Tsukuba Central 6, 1-1, Higashi 1-chome,Tsukuba-shi, Ibaraki-ken 305-8566, Japan, and they are described in U.S.Pat. No. 6,586,562.

It should be noted that the culture of microorganisms in the presentinvention, the production of PHA by microorganisms and accumulation inthe cells in the present invention, and the recovery of PHA from thecells in the present invention are not limited to the above describedmethods.

The composition of an inorganic salt culture medium (M9 medium) used inone method of the present invention is shown below.

[M9 medium]

-   Na₂HPO₄: 6.3-   KH₂PO₄: 3.0-   NH₄Cl: 1.0-   NaCl: 0.5 g/L, pH 7.0

To ensure satisfactory growth of cells and associated good productivityof PHA, it is necessary to add an approximately 0.3% (v/v) tracecomponent solution shown below to the above inorganic salt medium.

[Trace Component Solution]

-   nitrilotriacetic acid: 1.5; MgSO₄: 3.0; MnSO₄: 0.5;-   NaCl: 1.0; FeSO₄: 0.1;-   CaCl₂: 0.1; CoCl₂: 0.1; ZnSO₄: 0.1; CuSO₄: 0.1;-   AlK(SO₄)₂: 0.1, H₃BO₃: 0.1; Na₂MoO₄: 0.1; NiCl₂: 0.1 g/L

EXAMPLES Example 1

A Pseudomonas cichorii YN2 strain was inoculated into 200 mL of M9medium containing 0.5% D-glucose, 0.1% polypeptone and 0.1%5-[(phenylmethyl)oxy]valeric acid, followed by shaking the medium at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation, and they were resuspended in 200 mL of M9 mediumcontaining 0.5% D-glucose and 0.1% 5-[(phenylmethyl)oxy]valeric acid,followed by further shaking the resulting liquid at 30° C. at 125strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 33 mg of PHA.

An NMR analysis was carried out on the obtained PHA under the followingconditions:

-   <Measuring equipment> FT-NMR: Bruker DPX400-   Resonance frequency: ¹H=400 MHz-   <Measuring equipment> Type of nuclear species: ¹H-   Used solvent: CDCl₃-   Measuring temperature: room temperature

FIG. 1 shows a ¹H-NMR spectrum chart, and Table 1 shows theidentification results. TABLE 1 Chemical shift Integration (ppm)Attribution Fragmentation ratio 1.86 d1 m 2H 2.54 b1 m 2H 3.44 e1 m 2H4.41 f1 s 2H 5.31 c1 m 1H 7.20 to 7.31 h1 i1 j1 k1 l1 m 5H

As shown in Table 1, it was found that the obtained PHA was a PHAexpressed by chemical formula (26), which comprised3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit andfurther comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 94.9 mol %3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit.

Furthermore, the molecular weight of the obtained PHA was evaluated bygel permeation chromatography (GPC: Tosoh HLC-8220, column: TosohTSK-GEL Super HM-H, solvent: chloroform, polystyrene conversion). As aresult, Mn=123,000, and Mw=293,000.

Example 2

A Pseudomonas cichorii YN2 strain was inoculated into 200 mL of M9medium containing 0.5% D-glucose and 0.1% 5-[(phenylmethyl)oxy]valericacid, followed by shaking the medium at 30° C. at 125 strokes/minute. At48 hours later, cells were recovered by centrifugation, and they wereresuspended in 200 mL of M9 medium, which contained 0.5% D-glucose and0.1% 5-[(phenylmethyl)oxy]valeric acid but did not contain a nitrogensource (NH₄Cl), followed by further shaking the resulting liquid at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 30 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAwas a PHA expressed by chemical formula (26), which comprised3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit andfurther comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 92.6 mol %3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit.

Example 3

A Pseudomonas cichorii H45 strain was inoculated into 200 mL of M9medium containing 0.5% D-glucose, 0.1% polypeptone, and 0.1%5-[(phenylmethyl)oxy]valeric acid, followed by shaking the medium at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation, and they were resuspended in 200 mL of M9 mediumcontaining 0.5% D-glucose and 0.1% 5-[(phenylmethyl)oxy]valeric acid,followed by further shaking the resulting liquid at 30° C. at 125strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 31 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAwas a PHA expressed by chemical formula (26), which comprised3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit andfurther comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidfatty acid having 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 91.6 mol %3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit.

Example 4

A Pseudomonas jessenii P161 strain was inoculated into 200 mL of M9medium containing 0.5% D-glucose, 0.1% polypeptone, and 0.1%5-[(phenylmethyl)oxy]valeric acid, followed by shaking the medium at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation, and they were resuspended in 200 mL of M9 mediumcontaining 0.5% D-glucose and 0.1% 5-[(phenylmethyl)oxy]valeric acid,followed by further shaking the resulting liquid at 30° C. at 125strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 29 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAwas a PHA expressed by chemical formula (26), which comprised3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit andfurther comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 90.8 mol %3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit.

Example 5

A Pseudomonas cichorii YN2 strain was inoculated into 200 mL of M9medium containing 0.5% D-glucose, 0.1% polypeptone, and 0.1%5-[(phenylmethyl)oxy]valeric acid, followed by shaking the medium at 30°C. at 125 strokes/minute. At 48 hours later, 20 mL of aqueous solutioncontaining 5% D-glucose and 1% 5-[(phenylmethyl)oxy]valeric acid wasadded thereto, followed by further shaking the resulting liquid at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 25 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAwas a PHA expressed by chemical formula (26), which comprised3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit andfurther comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 79.7 mol %3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit.

Example 6

A Pseudomonas cichorii YN2 strain was inoculated into 200 mL of M9medium containing 0.5% polypeptone and 0.1% 5-[(phenylmethyl)oxy]valericacid, followed by shaking the medium at 30° C. at 125 strokes/minute. At48 hours later, cells were recovered by centrifugation, and they wereresuspended in 200 mL of M9 medium, which contained 0.5% pyruvic acidand 0.1% 5-[(phenylmethyl)oxy]valeric acid but did not contain anitrogen source (NH₄Cl), followed by further shaking the resultingliquid at 30° C. at 125 strokes/minute. At 48 hours later, cells wererecovered by centrifugation and washed once with cold methanol, and thenlyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 24 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAwas a PHA expressed by chemical formula (26), which comprised3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit andfurther comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 77.8 mol %3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit.

Example 7

A Pseudomonas cichorii YN2 strain was inoculated into 200 mL of M9medium containing 0.5% polypeptone and 0.1% 5-[(phenylmethyl)oxy]valericacid, followed by shaking the medium at 30° C. at 125 strokes/minute. At48 hours later, fungus cells were recovered by centrifugation and washedonce with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 20 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAwas a PHA expressed by chemical formula (26), which comprised3-hydroxy-5[(phenylmethyl)oxy]valeric acid as a monomer unit and furthercomprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 74.2 mol %3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit.

Example 8

A Pseudomonas cichorii H45 strain was inoculated into 200 mL of M9medium containing 0.5% yeast extract and 0.1%5-[(phenylmethyl)oxy]valeric acid, followed by shaking the medium at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 660° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 16 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAwas a PHA expressed by chemical formula (26), which comprised3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit andfurther comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 75.9 mol %3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit.

Example 9

A Pseudomonas jessenii P161 strain was inoculated into 200 mL of M9medium containing 0.5% glucose and 0.1% 5-[(phenylmethyl)oxy]valericacid, followed by shaking the medium at 30° C. at 125 strokes/minute. At48 hours later, cells were recovered by centrifugation and washed oncewith cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 17 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAwas a PHA expressed by chemical formula (26), which comprised3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit andfurther comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 81.5 mol %3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit.

Example 10

A Pseudomonas cichorii YN2 strain was inoculated into 200 mL of M9medium containing 0.5% pyruvic acid and 0.1%5-[(phenylmethyl)oxy]valeric acid, followed by shaking the medium at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 10 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAwas a PHA expressed by chemical formula (26), which comprised3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit andfurther comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 88.5 mol %3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit.

Example 11

A Pseudomonas cichorii H45 strain was inoculated into 200 mL of M9medium containing 0.5% sodium glutamate and 0.1%5-[(phenylmethyl)oxy]valeric acid, followed by shaking the medium at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 12 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAwas a PHA expressed by chemical formula (26), which comprised3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit andfurther comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 86.3 mol %3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit.

Example 12

A Pseudomonas jessenii P161 strain was inoculated into 200 mL of M9medium containing 0.1% nonanoic acid and 0.1%5-[(phenylmethyl)oxy]valeric acid, followed by shaking the medium at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 9 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAwas a PHA expressed by chemical formula (26), which comprised3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit andfurther comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 24.5 mol %3-hydroxy-5-[(phenylmethyl)oxy]valeric acid as a monomer unit.

Example 13

A Pseudomonas cichorii YN2 strain was inoculated into 200 mL of M9medium containing 0.5% D-glucose, 0.1% polypeptone, and 0.1%4-[(phenylmethyl)oxy]butyric acid, followed by shaking the medium at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation, and they were resuspended in 200 mL of M9 mediumcontaining 0.5% D-glucose and 0.1% 4-[(phenylmethyl)oxy]butyric acid,followed by further shaking the resulting liquid at 30° C. at 125strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 30 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAcomprised 3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unitand further comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 92.4 mol %3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unit.

Furthermore, the molecular weight of the obtained PHA was evaluated bygel permeation chromatography (GPC: Tosoh HLC-8220, column: TosohTSK-GEL Super HM-H, solvent: chloroform, polystyrene conversion). As aresult, Mn=138,000, and Mw 294,000.

Example 14

A Pseudomonas cichorii YN2 strain was inoculated into 200 mL of M9medium containing 0.5% D-glucose and 0.1% 4-[(phenylmethyl)oxy]butyricacid, followed by shaking the medium at 30° C. at 125 strokes/minute. At48 hours later, cells were recovered by centrifugation, and they wereresuspended in 200 mL of M9 medium, which contained 0.5% D-glucose and0.1% 4-[(phenylmethyl)oxy]butyric acid but did not contain a nitrogensource (NH₄Cl), followed by further shaking the resulting liquid at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 26 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAcomprised 3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unitand further comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 90.5 mol %3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unit.

Example 15

A Pseudomonas cichorii YN2 strain was inoculated into 200 mL of M9medium containing 0.5% D-glucose, 0.1% polypeptone, and 0.1%4-[(phenylmethyl)oxy]butyric acid, followed by shaking the medium at 30°C. at 125 strokes/minute. At 48 hours later, 20 mL of aqueous solutioncontaining 5% D-glucose and 1% 4-[(phenylmethyl)oxy]butyric acid wasadded thereto, followed by further shaking the resulting liquid at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 20 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAcomprised 3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unitand further comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 76.8 mol %3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unit.

Example 16

A Pseudomonas cichorii YN2 strain was inoculated into 200 mL of M9medium containing 0.5% polypeptone and 0.1% 4-[(phenylmethyl)oxy]butyricacid, followed by shaking the medium at 30° C. at 125 strokes/minute. At48 hours later, cells were recovered by centrifugation, and they wereresuspended in 200 mL of M9 medium, which contained 0.5% pyruvic acidand 0.1% 4-[(phenylmethyl)oxy]butyric acid but did not contain anitrogen source (NH₄Cl), followed by further shaking the resultingliquid at 30° C. at 125 strokes/minute. At 48 hours later, cells wererecovered by centrifugation and washed once with cold methanol, and thenlyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 19 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAcomprised 3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unitand further comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 73.2 mol %3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unit.

Example 17

A Pseudomonas cichorii YN2 strain was inoculated into 200 mL of M9medium containing 0.5% polypeptone and 0.1% 4-[(phenylmethyl)oxy]butyricacid, followed by shaking the medium at 30° C. at 125 strokes/minute. At48 hours later, cells were recovered by centrifugation and washed oncewith cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 15 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAcomprised 3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unitand further comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 76.7 mol %3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unit.

Example 18

A Pseudomonas cichorii H45 strain was inoculated into 200 mL of M9medium containing 0.5% yeast extract and 0.1%4-[(phenylmethyl)oxy]butyric acid, followed by shaking the medium at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 14 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAcomprised 3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unitand further comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 75.5 mol %3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unit.

Example 19

A Pseudomonas jessenii P161 strain was inoculated into 200 mL of M9medium containing 0.5% glucose and 0.1% 4-[(phenylmethyl)oxy]butyricacid, followed by shaking the medium at 30° C. at 125 strokes/minute. At48 hours later, cells were recovered by centrifugation and washed oncewith cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 11 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAcomprised 3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unitand further comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 85.9 mol %3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unit.

Example 20

A Pseudomonas cichorii YN2 strain was inoculated into 200 mL of M9medium containing 0.5% pyruvic acid and 0.1%4-[(phenylmethyl)oxy]butyric acid, followed by shaking the medium at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 8 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAcomprised 3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unitand further comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 90.4 mol %3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unit.

Example 21

A Pseudomonas cichorii H45 strain was inoculated into 200 mL of M9medium containing 0.5% sodium glutamate and 0.1%4-[(phenylmethyl)oxy]butyric acid, followed by shaking the medium at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 10 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAcomprised 3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unitand further comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 84.3 mol %3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unit.

Example 22

A Pseudomonas jessenii P161 strain was inoculated into 200 mL of M9medium containing 0.1% nonanoic acid and 0.1%4-[(phenylmethyl)oxy]butyric acid, followed by shaking the medium at 30°C. at 125 strokes/minute. At 48 hours later, cells were recovered bycentrifugation and washed once with cold methanol, and then lyophilized.

The obtained lyophilized pellet was suspended in 20 mL of chloroform,and the suspension was stirred at 60° C. for 20 hours to extract PHA.The extract was filtered through a membrane filter with a pore size of0.45 μm, and the filtrate was concentrated using a rotary evaporator.The obtained condensate was reprecipitated in cold methanol, and thenonly the precipitate was recovered and subjected to vacuum drying, toobtain 7 mg of PHA. As a result of carrying out an NMR analysis underthe same conditions as in Example 1, it was found that the obtained PHAcomprised 3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unitand further comprised, as a monomer unit, 3-hydroxyalkanoic acid or3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid. Moreover, from the ¹H-NMR spectrum integrationratio, it was found that the obtained PHA comprised 21.9 mol %3-hydroxy-4-[(phenylmethyl)oxy]butyric acid as a monomer unit.

Example 23

0.5% glucose, 6 mM 5-phenoxyvaleric acid, and 3 mM5-[(phenylmethyl)oxy]valeric acid were dissolved in 100 ml of the aboveM9 medium, and the resultant solution was placed in a 200 ml shakingflask and was then sterilized with an autoclave, followed by cooling toroom temperature. 2 ml of the culture solution of a Pseudomonas cichoriiYN2 strain that had previously been subjected to shaking culture at 30°C. for 8 hours in an M9 medium containing 0.5% polypeptone was added tothe above prepared medium, followed by culture at 30° C. for 48 hours.After completion of the culture, cells were recovered, and the thusobtained cells were resuspended in 100 ml of the same above medium,followed by culture in a 200 ml shaking flask at 30° C. for 42 hours.After completion of the culture, cells were recovered by centrifugationand washed with methanol, and then dried. After weighing the driedcells, chloroform was added thereto, and the mixture was stirred at 35°C. for 72 hours, to extract a polymer. The chloroform containing theextracted polymer was filtered, and the filtrate was concentrated usingan evaporator. Thereafter, the precipitated and solidified portion wascollected in cold methanol, and the portion was dried under reducedpressure, to obtain a polymer of interest. FIG. 2 shows the resultsobtained from an NMR analysis that was carried out under the sameconditions as in Example 1. It was confirmed that the obtained PHA was apolyhydroxyalkanoate copolymer comprising the units expressed by thefollowing chemical formula (27) (A:B:other units (3-hydroxyalkanoic acidor 3-hydroxyalkenoic acid, which is saturated or unsaturated fatty acidhaving 4 to 12 carbon atoms, such as 3-hydroxybutyric acid or3-hydroxyvaleric acid)=63:37:0). Moreover, it was confirmed by ¹³C-NMR(<measuring equipment> FT-NMR: Bruker DP×400, resonance frequency:¹³C=100 MHz, <measuring equipment> type of nuclear species: ¹³C, usedsolvent: CDCl₃, measuring temperature: room temperature) that theobtained PHA comprised unit B, that is, a3-hydroxy-5-[(phenylmethyl)oxy]valeric acid unit.

The molecular weight of the polymer was determined by gel permeationchromatography (GPC) (GPC: Tosoh HLC-8220, column: Tosoh TSK-GEL SuperHM-H, solvent: chloroform, polystyrene conversion).

The weight of the obtained polymer (PDW) was 0.17 g/l, and the numberaverage molecular weight of the obtained polymer was 93,000.

Example 24

0.5% glucose, 0.1% polypeptone, 6 mM 5-phenoxyvaleric acid, and 3 mM5-[(phenylmethyl)oxy]valeric acid were dissolved in 100 ml of the aboveM9 medium, and the resultant solution was placed in a 200 ml shakingflask and was then sterilized with an autoclave, followed by cooling toroom temperature. 2 ml of the culture solution of a Pseudomonas cichoriiYN2 strain that had previously been subjected to shaking culture at 30°C. for 8 hours in an M9 medium containing 0.5% polypeptone was added tothe above prepared medium, followed by culture at 30° C. for 42 hours.After completion of the culture, cells were recovered and washed withmethanol, and then dried. After weighing the dried cells, chloroform wasadded thereto, and the mixture was stirred at 35° C. for 72 hours, toextract a polymer. The chloroform containing the extracted polymer wasfiltered, and the filtrate was concentrated using an evaporator.Thereafter, the precipitated and solidified portion was collected incold methanol, and the portion was dried under reduced pressure, toobtain a polymer of interest.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (27)(A:B:other units (3-hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=38:33:29). Moreover, by ¹³C-NMR that was carried out under thesame condition as in Example 23, it was confirmed that the obtainedpolymer comprised unit B, that is, a3-hydroxy-5-[(phenylmethyl)oxy]valeric acid unit.

The molecular weight of the polymer was determined by GPC in the samemanner as in Example 1.

The weight of the obtained polymer (PDW) was 0.06 g/l, and the numberaverage molecular weight of the obtained polymer was 94,000.

Example 25

A polymer of interest was obtained by the same method as in Example 24with the exceptions that a Pseudomonas cichorii H45 strain was usedinstead of the YN2 strain used in Example 24, and that 0.5% yeastextract was used instead of glucose and polypeptone used in Example 24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (27)(A:B:other units (3-hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=42:33:25). Moreover, ¹³C-NMR was carried out in the same manner asin Example 23, and as a result, it was confirmed that the obtainedpolymer comprised unit B, that is, a3-hydroxy-5-[(phenylmethyl)oxy]valeric acid unit.

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.05 g/l, and the numberaverage molecular weight of the obtained polymer was 91,000.

Example 26

A polymer of interest was obtained by the same method as in Example 24with the exceptions that a Pseudomonas cichorii H45 strain was usedinstead of the YN2 strain used in Example 24, and that 0.5% sodiumpyruvate was used instead of glucose and polypeptone used in Example 24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (27)(A:B:other units (3-hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=58:24:18). Moreover, ¹³C-NMR was carried out in the same manner asin Example 23, and as a result, it was confirmed that the obtainedpolymer comprised unit B, that is, a3-hydroxy-5-[(phenylmethyl)oxy]valeric acid unit.

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.03 g/l, and the numberaverage molecular weight of the obtained polymer was 102,000.

Example 27

A polymer of interest was obtained by the same method as in Example 24with the exceptions that a Pseudomonas jessenii P161 strain was usedinstead of the YN2 strain used in Example 24, and that 0.5% sodiumglutamate was used instead of glucose and polypeptone used in Example24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (27)(A:B:other units (3-hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=40:35:25). Moreover, ¹³C-NMR was carried out in the same manner asin Example 23, and as a result, it was confirmed that the obtainedpolymer comprised unit B, that is, a3-hydroxy-5-[(phenylmethyl)oxy]valeric acid unit.

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.08 g/l, and the numberaverage molecular weight of the obtained polymer was 89,000.

Example 28

A polymer of interest was obtained by the same method as in Example 24with the exceptions that a Pseudomonas jessenii P161 strain was usedinstead of the YN2 strain used in Example 24, and that 0.1% nonanoicacid was used instead of glucose and polypeptone used in Example 24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (27)(A:B:other units=40:35:25). Moreover, ¹³C-NMR was carried out in thesame manner as in Example 23, and as a result, it was confirmed that theobtained polymer comprised unit B, that is, a3-hydroxy-5-[(phenylmethyl)oxy]valeric acid unit.

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.04 g/l, and the numberaverage molecular weight of the obtained polymer was 98,000.

Example 29

A polymer of interest was obtained by the same method as in Example 24with the exception that 4-phenoxybutyric acid was used instead of5-phenoxyvaleric acid used in Example 24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (28)(A:B:other units (3-hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=21:43:36). Moreover, ¹³C-NMR measurement was carried out in thesame manner as in Example 23, and as a result, it was confirmed that theobtained polymer comprised unit B, that is, a3-hydroxy-5-[(phenylmethyl)oxy]valeric acid unit.

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.02 g/l, and the numberaverage molecular weight of the obtained polymer was 92,000.

Example 30

A polymer of interest was obtained by the same method as in Example 24with the exception that 5-phenylvaleric acid was used instead of5-phenoxyvaleric acid used in Example 24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (29)(A:B:other units (3-hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=56:25:19).

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.13 g/l, and the numberaverage molecular weight of the obtained polymer was 98,000.

Example 31

A polymer of interest was obtained by the same method as in Example 24with the exception that 5-(4-vinylphenyl)valeric acid was used insteadof 5-phenoxyvaleric acid used in Example 24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (30)(A:B:other units (3-hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=42:34:24).

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.03 g/l, and the numberaverage molecular weight of the obtained polymer was 87,000.

Example 32

A polymer of interest was obtained by the same method as in Example 24with the exception that 5-benzoylvaleric acid was used instead of5-phenoxyvaleric acid used in Example 24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (31)(A:B:other units (3-hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=48:27:25).

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.02 g/l, and the numberaverage molecular weight of the obtained polymer was 160,000.

Example 33

A polymer of interest was obtained by the same method as in Example 24with the exception that 5-(phenylsulfanyl)valeric acid was used insteadof 5-phenoxyvaleric acid used in Example 24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (32)(A:B:other units (3-hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=56:22:22).

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.11 g/l, and the numberaverage molecular weight of the obtained polymer was 89,000.

Example 34

A polymer of interest was obtained by the same method as in Example 24with the exception that 5-[(phenylmethyl)sulfanyl]valeric acid was usedinstead of 5-phenoxyvaleric acid used in Example 24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (33)(A:B:other units (3-hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=46:31:24).

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.04 g/l, and the numberaverage molecular weight of the obtained polymer was 84,000.

Example 35

A polymer of interest was obtained by the same method as in Example 24with the exception that 5-(2-thienyl)valeric acid was used instead of5-phenoxyvaleric acid used in Example 24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (34)(A:B:other units (3-hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=51:26:23).

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.09 g/l, and the numberaverage molecular weight of the obtained polymer was 86,000.

Example 36

A polymer of interest was obtained by the same method as in Example 24with the exception that 5-(2-thienylsulfanyl)valeric acid was usedinstead of 5-phenoxyvaleric acid used in Example 24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (35)(A:B:other units (3-hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=49:40:11).

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.10 g/l, and the numberaverage molecular weight of the obtained polymer was 81,000.

Example 37

A polymer of interest was obtained by the same method as in Example 24with the exception that 5-(2-thienylcarbonyl)valeric acid was usedinstead of 5-phenoxyvaleric acid used in Example 24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (36)(A:B:other units (3-hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=41:40:19).

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.02 g/l, and the numberaverage molecular weight of the obtained polymer was 89,000.

Example 38

A polymer of interest was obtained by the same method as in Example 24with the exception that 5-cyclohexylvaleric acid was used instead of5-phenoxyvaleric acid used in Example 24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (37)(A:B:other units (3-hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=46:28:26).

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.08 g/l, and the numberaverage molecular weight of the obtained polymer was 92,000.

Example 39

A polymer of interest was obtained by the same method as in Example 24with the exception that 4-[(phenylmethyl)oxy]butyric acid was usedinstead of 5-[(phenylmethyl)oxy]valeric acid used in Example 24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (38)(A:B:other units (3-hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=49:24:27). Moreover, ¹³C-NMR measurement was carried out in thesame manner as in Example 23, and as a result, it was confirmed that theobtained polymer comprised unit B, that is, a3-hydroxy-4-[(phenylmethyl)oxy]butyric acid unit.

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.02 g/l, and the numberaverage molecular weight of the obtained polymer was 91,000.

Example 40

A polymer of interest was obtained by the same method as in Example 24with the exception that 3 mM 5-phenoxyvaleric acid and 3 mM5-cyclohexylvaleric acid were used instead of 6 mM 5-phenoxyvaleric acidused in Example 24.

To determine the structure of the obtained polymer, ¹H-NMR was carriedout in the same manner as in Example 1. As a result, it was confirmedthat the obtained polymer was a polyhydroxyalkanoate copolymercomprising the units expressed by the following chemical formula (39)(A:B:C:other units (3hydroxyalkanoic acid or 3-hydroxyalkenoic acid,which is saturated or unsaturated fatty acid having 4 to 12 carbonatoms, such as 3-hydroxybutyric acid or 3-hydroxyvalericacid)=31:28:21:20). Moreover, ¹³C-NMR measurement was carried out in thesame manner as in Example 23, and as a result, it was confirmed that theobtained polymer comprised unit C, that is, a3-hydroxy-5-[(phenylmethyl)oxy]valeric acid unit.

As with Example 1, the molecular weight of the obtained polymer wasdetermined by GPC.

The weight of the obtained polymer (PDW) was 0.09 g/l, and the numberaverage molecular weight of the obtained polymer was 93,000.

1. A polyhydroxyalkanoate comprising a3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acid unit expressed by chemicalformula (1):

wherein x can be one or more integers within the range shown in thechemical formula.
 2. The polyhydroxyalkanoate according to claim 1,comprising at least one unit expressed by chemical formula selected fromthe group consisting of chemical formulas (2) and (3):

wherein y and z can be one or more integers within the range shown inthe chemical formulas, while being independent from the unit expressedby chemical formula (1).
 3. The polyhydroxyalkanoate according to claim1, comprising, in a molecule thereof, the3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acid unit expressed by chemicalformula (1) and a 3-hydroxy-alkanoic acid unit expressed by chemicalformula (4):

wherein m can be one or more integers within the range shown in thechemical formula, and R comprises a residue having either a phenylstructure or thienyl structure, or a 3-hydroxy-ω-cyclohexylalkanoic acidunit expressed by chemical formula (5):

wherein R₁ is H, CN, NO₂, halogen, CH₃, C₂H₅, C₃H₇, CF₃, C₂F₅ and C₃F₇,and k can be one or more integers within the range shown in the chemicalformula.
 4. The polyhydroxyalkanoate according to claim 1, wherein the3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acid unit expressed by chemicalformula (1) is either one or both of: a3-hydroxy-4-[(phenylmethyl)oxy]butyric acid unit expressed by chemicalformula (6):

and a 3-hydroxy-5-[(phenylmethyl)oxy]valeric acid unit expressed bychemical formula (7):


5. The polyhydroxyalkanoate according to claim 3, wherein R in chemicalformula (4) is a group selected from the group consisting of

wherein R₂ is H, halogen, CN, NO₂, CH₃, C₂H₅, C₃H₇, CH═CH₂, COOR₃(wherein R₃ represents any one selected from the group consisting of H,Na and K), CF₃, C₂F₅ and C₃F₇, and in a case where there exist aplurality of units, R₂ may be different for each unit;

wherein R₄ is selected from the group consisting of H, halogen, CN, NO₂,CH₃, C₂H₅, C₃H₇, SCH₃, CF₃, C₂F₅ and C₃F₇, and in a case where thereexist a plurality of units, R₄ may be different for each unit;

wherein R₅ is selected from the group consisting of H, halogen, CN, NO₂,CH₃, C₂H₅, C₃H₇, CF₃, C₂F₅ and C₃F₇, and in a case where there exist aplurality of units, R₅ may be different for each unit;

wherein R₆ is selected from the group consisting of H, halogen, CN, NO₂,COOR₇, SO₂R₈ (wherein R₇ represents any one selected from the groupconsisting of H, Na, K, CH₃ and C₂H₅, and Re represents any one selectedfrom the group consisting of OH, ONa, OK, halogen, OCH₃ and OC₂H₅), CH₃,C₂H₅, C₃H₇, (CH₃)₂—CH, and (CH₃)₃—C, and in a case where there exist aplurality of units, R₆ may be different for each unit;

wherein R₉ represents a substituent group on the aromatic ring, R₉ isselected from thg group consisting of H, halogen, CN, NO₂, COOR₁₀,SO₂R₁₁ (wherein R₁₀ represents any one selected from the groupconsisting of H, Na, K, CH₃ and C₂H₅, and R₁₁ represents any oneselected from the group consisting of OH, ONa, OK, halogen, OCH₃ andOC₂H₅), CH₃, C₂H₅, C₃H₇, (CH₃)₂—CH and (CH₃)₃—C, and in a case wherethere exist a plurality of units, R₉ may be different for each unit;

wherein R₁₂ is selected from thg group consisting of H, halogen, CN,NO₂, COOR₁₃, SO₂R₁₄ (wherein R₁₃ represents any one selected from thegroup consisting of H, Na, K, CH₃ and C₂H₅, and R₁₄ represents any oneselected from the group consisting of OH, ONa, OK, halogen, OCH₃ andOC₂H₅), CH₃, C₂H₅, C₃H₇, (CH₃)₂—CH and (CH₃)₃—C, and in a case wherethere exist a plurality of units, R₁₂ may be different for each unit;and

wherein R₁₅ is selected from the group consisting of H, halogen, CN,NO₂, COOR₁₆, SO₂R₁₇ (wherein R₁₆ represents any one selected from thegroup consisting of H, Na, K, CH₃ and C₂H₅, and R₁₇ represents any oneselected from the group consisting of OH, ONa, OK, halogen, OCH₃ andOC₂H₅), CH₃, C₂H₅, C₃H₇, (CH₃)₂—CH and (CH₃)₃—C, and in a case wherethere exist a plurality of units, R₁₅ may be different for each unit. 6.The polyhydroxyalkanoate according to claim 1, wherein a number averagemolecular weight is within the range between 1,000 and 1,000,000.
 7. Amethod for producing a polyhydroxyalkanoate comprising, in a moleculethereof, a 3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acid unit expressedby chemical formula (1):

wherein x can be one or more integers within the range shown in thechemical formula, which comprises allowing a microorganism with anability to produce a polyhydroxyalkanoate comprising in a moleculethereof a 3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acid unit expressed bychemical formula (1) to biosynthesize the polyhydroxyalkanoate under acondition which comprise ω-[(phenylmethyl)oxy]alkanoic acid expressed bychemical formula (19):

wherein x can be one or more integers within the range shown in thechemical formula.
 8. The method for producing a polyhydroxyalkanoateaccording to claim 7, wherein the polyhydroxyalkanoate comprises atleast one unit expressed by the following chemical formulas (2) and (3):

wherein y and z can be one or more integers within the range shown inthe chemical formulas, while being independent from the unit expressedby chemical formula (1).
 9. The method for producing apolyhydroxyalkanoate according to claim 7, comprising allowing themicroorganism with an ability to produce a polyhydroxyalkanoatecomprising, in a molecule thereof, the3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acid unit expressed by chemicalformula (1) and a 3-hydroxy-alkanoic acid unit expressed by chemicalformula (22):

wherein m can be one or more integers within the range shown in thechemical formula, and R₁₈ comprises a residue having either a phenylstructure or thienyl structure, or 3-hydroxy-ω-cyclohexylalkanoic acidunit expressed by chemical formula (5):

wherein R₁ is selected from the group consisting of H, CN, NO₂, halogen,CH₃, C₂H₅, C₃H₇, CF₃, C₂F₅ and C₃F₇, and k can be one or more integerswithin the range shown in the chemical formula, fromω-[(phenylmethyl)oxy]alkanoic acid expressed by chemical formula (19),and a alkanoic acid expressed by chemical formula (20):

wherein q can be one or more integers within the range shown in thechemical formula, and R₁₆ comprises a residue having either a phenylstructure or thienyl structure, or ω-cyclohexylalkanoic acid expressedby chemical formula (21):

wherein R₁₇ is selected from the group consisting of H, CN, NO₂,halogen, CH₃, C₂H₅, C₃H₇, CF₃, C₂F₅ and C₃F₇, and r can be one or moreintegers within the range shown in the chemical formula as raw materialsto biosynthesize the polyhydroxyalkanoate under a condition whichcomprise ω-[(phenylmethyl)oxy]alkanoic acid expressed by chemicalformula (19), and alkanoic acid expressed by chemical formula (20) orω-cyclohexylalkanoic acid expressed by chemical formula (21).
 10. Themethod for producing a polyhydroxyalkanoate according to claim 7,wherein the ω-[(phenylmethyl)oxy]alkanoic acid expressed by saidchemical formula (19) is 4-[(phenylmethyl)oxy]butyric acid expressed bychemical formula (23):

or 5-[(phenylmethyl)oxy]valeric acid expressed by chemical formula (24):


11. The method for producing a polyhydroxyalkanoate according to claim9, wherein R₁₆ in chemical formula (20) and R₁₈ in chemical formula (22)are groups independently selected from the group consisting of

wherein R₁₉ is selected from the group consisting of H, halogen, CN,NO₂, CH₃, C₂H₅, C₃H₇, CH═CH₂, CF₃, C₂F₅ and C₃F₇, and in a case wherethere exist a plurality of units, R₁₉ may be different for each unit;

wherein R₄ is selected from the group consisting of H, halogen, CN, NO₂,CH₃, C₂H₅, C₃H₇, SCH₃, CF₃, C₂F₅ and C₃F₇, and in a case where thereexist a plurality of units, R₄ may be different for each unit;

wherein R₅ is selected from the group consisting of H, halogen, CN, NO₂,CH₃, C₂H₅, C₃H₇, CF₃, C₂F₅ and C₃F₇, and in a case where there exist aplurality of units, R₅ may be different for each unit;

wherein R₆ is selected from the group consisting of H, halogen, CN, NO₂,COOR₇, SO₂R₈ (wherein R₇ represents any one selected from the groupconsisting of H, Na, K, CH₃ and C₂H₅, and R₈ represents any one selectedfrom the group consisting of OH, ONa, OK, halogen, OCH₃ and OC₂H₅), CH₃,C₂H₅, C₃H₇, (CH₃)₂—CH and (CH₃)₃—C, and in a case where there exist aplurality of units, R₆ may be different for each unit;

wherein R₉ is selected from the group consisting of H, halogen, CN, NO₂,COOR₁, SO₂R₁₁ (wherein R₁₀ represents any one selected from the groupconsisting of H, Na, K, CH₃ and C₂H₅, and R₁₁ represents any oneselected from the group consisting of OH, ONa, OK, halogen, OCH₃ andOC₂H₅), CH₃, C₂H₅, C₃H₇, (CH₃)₂—CH and (CH₃)₃—C, and in a case wherethere exist a plurality of units, R₉ may be different for each unit;

wherein R₁₂ is selected from the group consisting of H, halogen, CN,NO₂, COOR₁₃, SO₂R₁₄ (wherein R₁₃ represents any one selected from thegroup consisting of H, Na, K, CH₃ and C₂H₅, and R₁₄ represents any oneselected from the group consisting of OH, ONa, OK, halogen, OCH₃ andOC₂H₅), CH₃, C₂H₅, C₃H₇, (CH₃)₂—CH and (CH₃)₃—C, and in a case wherethere exist a plurality of units, R₁₂ may be different for each unit;and

wherein R₁₅ is selected from the group consisting of H, halogen, CN,NO₂, COOR₁₆, SO₂R₁₇ (wherein R₁₆ represents any one selected from thegroup consisting of H, Na, K, CH₃ and C₂H₅, and R₁₇ represents any oneselected from the group consisting of OH, ONa, OK, halogen, OCH₃ andOC₂H₅), CH₃, C₂H₅, C₃H₇, (CH₃)₂—CH and (CH₃)₃—C, and in a case wherethere exist a plurality of units, R₁₅ may be different for each unit.12. The method for producing a polyhydroxyalkanoate according to claim7, wherein said condition is that said microorganisms is cultured in amedium containing ω-[(phenylmethyl)oxy]alkanoic acid expressed bychemical formula (19).
 13. The method for producing apolyhydroxyalkanoate according to claim 9, wherein said condition isthat said microorganism is cultured in a medium containing theω-[(phenylmethyl)oxy]alkanoic acid expressed by chemical formula (19)and the alkanoic acid expressed by chemical formula (20) or theω-cyclohexylalkanoic acid expressed by chemical formula (21).
 14. Themethod for producing a polyhydroxyalkanoate according to claim 12,wherein said medium contains at least one selected from the groupconsisting of peptides, yeast extract, organic acids or salts thereof,amino acids or salts thereof, saccharides and straight-chain alkanoicacids, which is saturated or unsaturated fatty acid having 4 to 12carbon atoms or salts thereof.
 15. The method for producing apolyhydroxyalkanoate according to claim 14, wherein the peptide ispolypeptone; the organic acids or salts thereof are one or morecompounds selected from the group consisting of pyruvic acid,oxaloacetic acid, citric acid, isocitric acid, ketoglutaric acid,succinic acid, fumaric acid, malic acid, lactic acid, and salts thereof;the amino acids or salts thereof are one or more compounds selected fromthe group consisting of glutamic acid, aspartic acid, and salts thereof;and the saccharides are one or more compounds selected from the groupconsisting of glyceroaldehyde, erythrose, arabinose, xylose, glucose,galactose, mannose, fructose, glycerol, erythritol, xylitol, gluconicacid, glucuronic acid and galacturonic acid, maltose, sucrose andlactose.
 16. The method for producing a polyhydroxyalkanoate accordingto claim 12, wherein said culture of microorganisms comprises two ormore culturing steps.
 17. The method for producing apolyhydroxyalkanoate according to claim 16, wherein said culture is afed-batch culture.
 18. The method for producing a polyhydroxyalkanoateaccording to any one of claims 12 to 17, comprising a step of recoveringa polyhydroxyalkanoate comprising3-hydroxy-ω-[(phenylmethyl)oxy]alkanoic acid unit expressed by chemicalformula (1) generated by the microorganism from the cells of themicroorganism.
 19. The method for producing a polyhydroxyalkanoateaccording to claim 7, wherein said microorganism belongs to Pseudomonasspecies.
 20. The method for producing a polyhydroxyalkanoate accordingto claim 19, wherein said microorganism is one or more strains selectedfrom the group consisting of Pseudomonas cichorii YN2 (FERM BP-7375),Pseudomonas cichorii H45 (FERM BP-7374) and Pseudomonas jessenii P161(FERN BP-7376).